Robert Aragon

Antibody-Mediated p53 Protein Therapy Prevents Liver Metastasis In vivo

To evaluate the clinical efficacy of monoclonal antibody (mAb) 3E10 Fv antibody-mediated p53 protein therapy, an Fv-p53 fusion protein produced in Pichia pastoris was tested on CT26.CL25 colon cancer cells in vitro and in vivo in a mouse model of colon cancer metastasis to the liver. In vitro experiments showed killing of CT26.CL25 cells by Fv-p53 but not Fv or p53 alone, and immunohistochemical staining confirmed that Fv was required for transport of p53 into cells. Prevention of liver metastasis in vivo was tested by splenic injection of 100 nmol/L Fv-p53 10 min and 1 week after injection of CT26.CL25 cancer cells into the portal vein of BALB/c mice. Mice were sacrificed 1 week after the second injection of Fv-p53 and assigned a quantitative metastasis score. Control mice had an average metastasis score of 3.3 +/- 1.3, whereas mice treated with Fv-p53 had an average metastasis score of 0.8 +/- 0.4 (P = 0.004). These results indicate that Fv-p53 treatment had a profound effect on liver metastasis and represent the first demonstration of effective full-length p53 protein therapy in vivo. mAb 3E10 Fv has significant clinical potential as a mediator of intracellular and intranuclear delivery of p53 for prevention and treatment of cancer metastasis.

RASSF1C modulates the expression of a stem cell renewal gene, PIWIL1.

BackgroundRASSF1A and RASSF1C are two major isoforms encoded by the Ras association domain family 1 (RASSF1) gene through alternative promoter selection and mRNA splicing. RASSF1A is a well established tumor suppressor gene. Unlike RASSF1A, RASSF1C appears to have growth promoting actions in lung cancer. In this article, we report on the identification of novel RASSF1C target genes in non small cell lung cancer (NSCLC).MethodsOver-expression and siRNA techniques were used to alter RASSF1C expression in human lung cancer cells, and Affymetrix-microarray study was conducted using NCI-H1299 cells over-expressing RASSF1C to identify RASSF1C target genes.ResultsThe microarray study intriguingly shows that RASSF1C modulates the expression of a number of genes that are involved in cancer development, cell growth and proliferation, cell death, and cell cycle. We have validated the expression of some target genes using qRT-PCR. We demonstrate that RASSF1C over-expression increases, and silencing of RASSF1C decreases, the expression of PIWIL1 gene in NSCLC cells using qRT-PCR, immunostaining, and Western blot analysis. We also show that RASSF1C over-expression induces phosphorylation of ERK1/2 in lung cancer cells, and inhibition of the MEK-ERK1/2 pathway suppresses the expression of PIWIL1 gene expression, suggesting that RASSF1C may exert its activities on some target genes such as PIWIL1 through the activation of the MEK-ERK1/2 pathway. Also, PIWIL1 expression is elevated in lung cancer cell lines compared to normal lung epithelial cells.ConclusionsTaken together, our findings provide significant data to propose a model for investigating the role of RASSF1C/PIWIL1 proteins in initiation and progression of lung cancer.

The small splice variant of HPV16 E6, E6, reduces tumor formation in cervical carcinoma xenografts.

High-risk types of human papillomavirus (HPV) cause nearly all cases of cervical cancer. The E6 oncoprotein is produced as a full-length variant (E6) as well as several shorter isoforms (E6). E6 inhibits certain oncogenic activities of E6, suggesting that it might play an anti-oncogenic role in vivo. To test this, we created E6-expressing SiHa (HPV(+)) and C33A (HPV(-)) cells, then examined the ability of both the parental and E6-expressing cells to form tumors in nude mice. We found that over-expression of E6 indeed decreased the growth of tumors derived from both SiHa and C33A cells, with the reduction greatest in tumors derived from E6-expressing SiHa cells. These findings point to multiple anti-oncogenic characteristics of E6, some of which likely involve down-regulation of the full-length isoform, and others that are independent of HPV. These data represent the first demonstration of biologically-relevant E6 activities distinct from those of the full-length isoform in vivo.

Ras-Association Domain Family 1C Protein Enhances Breast Tumor Growth In Vivo

The Ras association domain family 1 (RASSF1) gene is a Ras effector that plays an important role in carcinogenesis. We have previously shown that silencing of RASSF1C decreases and over-expression of RASSF1C increases cell proliferation, migration, and attenuates apoptosis of breast cancer cells in vitro. To further confirm our working hypothesis that RASSF1C may play a role as a growth promoter, we have tested the growth of human breast cancer cells stably over-expressing RASSF1A or RASSF1C in nude mice. Our studies show that breast cancer cells over-expressing HA-RASSF1A developed significantly smaller tumors and cells over-expressing HA-RASSF1C developed significantly larger tumors compared to control cells expressing the vector back bone. We have confirmed the expression of HA-RASSF1A and HA-RASSF1C in tumor tissue using RT-PCR, western blotting and immunohistochemical analyses using HA-antibody. Together, our previous in vitro and current in vivo findings further support our hypothesis that RASSF1C, unlike RASSF1A, is not a tumor suppressor and rather it appears to function as tumor growth promoter in breast cancer cells.

Abstract 1828: Proteomic analysis of the effect of E6 star expression on cellular pathways in HPV positive SiHa and HPV negative C33A cervical carcinoma cells

High-risk types of the human papillomavirus (HR-HPV) are the causative agents of nearly all cases of cervical cancer, as well as a significant number of head, neck, penile, vulvar and anal cancers. Like many other viruses with small genomes, HPV (∼8 kb) utilizes numerous mechanisms to increase the capacity of its genome to encode the proteins necessary for successful completion of its infectious life cycle, including alternative splicing. Studies over the past few decades have focused intensively on the activities and roles of E6 proteins from HR-HPVs during the process of cellular transformation, clearly implicating E6 as a major transforming agent. In contrast, the role of the smaller splice isoform, E6*, in the carcinogenic process has not yet been established. In a recent study, we demonstrated that the over-expression of E6* reduces tumor growth by SiHa (HPV16 positive) and C33A (no HPV) cells in nude mice, suggesting that therapies emulating the actions of E6* may be of medical benefit. Furthermore, tumor growth inhibition by E6* was greater in tumors derived from HPV positive cells than in tumors derived from HPV negative cells. This difference implies that E6* interferes with the oncogenic activity of the full-length protein as well as by acting through HPV-independent mechanisms. The goal of this study is to determine the pathways affected by E6* that may lead to the observed reduction in tumor formation in xenograft models. To elucidate how E6* may affect the levels of cellular proteins and thereby orchestrate pathway regulation, in both E6 positive and negative environments, SiHa pFlag, SiHa pE6*, C33A pFlag, and C33A pE6* cells were created and their differential protein expression examined using mass spectrometry and Ingenuity Pathway Analysis (IPA) software. Lysates of these cells were reduced, alkylated, trypsinized, and TMT labeled, and the labeled peptides were analyzed using an LTQ-Orbitrap Velos mass spectrometer. Proteins were quantified by TMT tags and identified by comparison against the human library using Proteome Discoverer Software. 322 proteins were detected as differentially expressed using a 1.3 fold-change cut-off value. Further analysis by IPA revealed that E6* induced changes in apoptosis and death receptor signaling pathways in both HPV- and HPV positive cells, while other pathways, such as those involving mitochondrial dysfunction and TNFR1 signaling, were more profoundly affected in HPV negative cells. Our study provides several promising leads for future experiments and analyses, specifically in the context of human cancers, and carries with it the exciting possibility of replicating the anti-oncogenic activity of E6* in such a way as to provide therapeutic benefit. Future work will involve more detailed examination of our preliminary results and comparing these observations with those obtained from actual tumors derived from these cells. Citation Format: Whitney Evans, Maria Filippova, Robert Aragon, Valeri Filippov, Mark E. Reeves, Penelope Duerksen-Hughes. Proteomic analysis of the effect of E6 star expression on cellular pathways in HPV positive SiHa and HPV negative C33A cervical carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1828. doi:10.1158/1538-7445.AM2015-1828

Abstract 3175: The splice variant of the human papillomavirus 16 E6 protein, E6*, displays anti-tumor properties in vivo

High-risk types of the human papillomavirus (HPV) are the causative agents of nearly all cases of cervical cancer, as well as a significant number of head, neck, penile, vulvar, and anal cancers. Like many other viruses with small genomes, HPV (∼8 kb) utilizes numerous mechanisms to increase the capacity of its genome to encode the proteins necessary for successful completion of its infectious life cycle, including alternative splicing. Studies over the past few decades have focused intensively on the activities and roles of E6 proteins from high-risk types of HPV during the process of cellular transformation, clearly implicating E6 as a major transforming agent. In contrast, the role of the smaller splice isoform, E6*, in the carcinogenic process (if any) has not yet been established. In the present study, we examined the behavior of the E6* protein during tumor growth in an in vivo nude mouse xenograft model. We created E6*-expressing SiHa (HPV+) and C33A (HPV-) cells, then examined the ability of both the parental and E6*-expressing cells to form tumors in nude mice. The difference in tumor size observed in the presence and absence of E6*, when expressed in both HPV+ (SiHa) and HPV- (C33A) cells, was dramatic and consistent, strongly indicating an anti-oncogenic role for E6* in this context. Interestingly, we found that tumor growth inhibition by E6* was greater in tumors derived from SiHa cells, which are HPV16-positive, than in tumors produced by C33A, which are HPV-negative. This difference implies that E6* acts by interfering with the oncogenic activity of the full-length protein as well as through one or more HPV-independent mechanisms. Consistent with this idea, we found that E6* does indeed bind to the full-sized isoform and inhibits its ability to accelerate degradation of p53 and procaspase 8. These data represent the first demonstration of biologically-relevant E6* activities distinct from those of the full-length isoform in vivo. The significance of these findings in the context of human cancers is in the possibility of mimicking or replicating the anti-oncogenic activity of E6* in such a way as to provide therapeutic benefit. Citation Format: Whitney Evans, Maria Filippova, Robert Aragon, Valeri Filippov, Mark Reeves, Penelope Duerksen-Hughes. The splice variant of the human papillomavirus 16 E6 protein, E6*, displays anti-tumor properties in vivo . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3175. doi:10.1158/1538-7445.AM2014-3175

Abstract 1139: Ras association domain family protein 1, isoform C (RASSF1C) promotes breast tumor growth in vivo

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Breast cancer will kill more than 40,000 women in the United States this year, second in cancer deaths only to carcinoma of the lung. Therefore, a better understanding of the molecules and pathways that lead to breast cancer development is important because it could lead to specific therapies that interrupt these pathways. Recently, the Ras association domain family 1 (RASSF1) gene was identified as a Ras effector that plays an important role in carcinogenesis. The RASSF1 gene encodes two major isoforms, RASSF1A and RASSF1C, derived by alternative promoter selection and alternative mRNA splicing. While RASSF1A is extensively studied and is an established tumor suppressor, very little is known about RASSF1C function Our laboratory is interested in determining the role of RASSF1C in human cancer cell growth. We have previously shown that of RASSF1C promotes cell proliferation and migration and attenuates apoptosis of breast cancer cells in vitro. To confirm our hypothesis that RASSF1C may be a growth promoter, we have tested the growth of human breast cancer cells over-expressing RASSF1C in nude mice. Our preliminary studies show that breast cancer cells over-expressing RASSF1C developed significantly larger tumors compared to control cells. We have confirmed the expression of RASSF1C in tumor tissue using both RT-PCR and immune-staining analyses. Together, our previous in vitro and current in vivo findings further support our hypothesis that RASSF1C, unlike RASSF1A, is not a tumor suppressor and rather it appears to play a role in stimulating proliferation and survival of breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1139. doi:10.1158/1538-7445.AM2011-1139